MPRAsnakeflow experiment QC report

Low correlation!

Spearman correlation across replicates is low using a barcode threshold per oligo of 5! Spearman correlation of DNA is 0.28, RNA is 0.58 and DNA/RNA ratio is 0.52. Minimum allowed values are 0.85, 0.95, and 0.75.

Overall quality metrics

Table explanation
  • median rna read count: Median of RNA read count for oligos that passed filtering to determine sufficient coverage in terms of read count. Value is the median of all replicates.
  • median barcodes passing filtering: Median number of barcodes across tested sequences that passed filtering to determine if there was sufficient barcode to oligo coverage. Value is the median of all replicates.
  • pearson correlation: The correlation of log2 RNA/DNA ratios across tested sequences as a measure of replicable activity signal. Value is the median of replicate comparisons using only oligos with >= 5 barcodes.
  • fraction oligos passing: Fraction of tested sequences that passed filtering of the mappable sequences to determine if the designed library was sufficiently recovered. Value is the median of all replicates and using only oligos with >= 5 barcodes.
median barcodes passing filtering median rna read count pearson correlation fraction oligos passing
10 48 0.67 0.82

DNA over RNA counts

Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected.

Oligo correlation

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 1563 oligos with a minimum number of 5 barcodes. Second tab shows all 2016 oligos that have assigned barcodes.

Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
HepG2 1 2 1660 1678 1560 0.32 0.60 0.53 0.32 0.70 0.62
HepG2 1 3 1660 1700 1553 0.28 0.61 0.55 0.28 0.71 0.63
HepG2 2 3 1678 1700 1578 0.31 0.58 0.52 0.31 0.68 0.62
Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
HepG2 1 2 2031 2029 2016 0.28 0.53 0.45 0.27 0.60 0.51
HepG2 1 3 2031 2030 2017 0.26 0.53 0.45 0.25 0.59 0.50
HepG2 2 3 2029 2030 2017 0.29 0.51 0.43 0.29 0.58 0.49

Experiment statistic

The total number of oligos in this experiment is 2050 (defined by the assignment) with 58369 associated barcodes.

In average across replicates we see 2030 from 25273 average barcodes in the count data and around 71252 barcodes where not in the assignment.

condition replicate oligos dna/rna matched barcodes unknown barcodes dna/rna % matched barcodes total dna counts total rna counts avg dna counts per bc avg rna counts per bc barcode outlier removed avg dna/rna barcodes per oligo
HepG2 1 2031 25015 71917 25.81 211999 566118 2.19 5.84 0 12.32
HepG2 2 2029 25431 71152 26.33 201596 564415 2.09 5.84 0 12.53
HepG2 3 2030 25372 70686 26.41 197128 602231 2.05 6.27 0 12.50
Experiment Barcodes Counts Average counts Assigned barcodes Assigned counts Average assigned counts Fraction assigned barcodes Fraction assigned counts
HepG2_1_DNA 149096 294140 1.97 32963 75033 2.28 0.22 0.26
HepG2_2_DNA 153331 289185 1.89 34276 74146 2.16 0.22 0.26
HepG2_3_DNA 152729 285381 1.87 34267 73476 2.14 0.22 0.26
HepG2_1_RNA 190734 871828 4.57 38348 229126 5.97 0.20 0.26
HepG2_2_RNA 187969 878320 4.67 38134 233299 6.12 0.20 0.27
HepG2_3_RNA 192775 959787 4.98 38566 253387 6.57 0.20 0.26

Histograms barcodes per oligo, counts per barcode

Histogramm of number of barcodes per oligo and the number of counts per barcode devidied by DNA and RNA. Median is red, mean is blue.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 1563 oligos with a minimum number of 5 barcodes. Second tab shows all 2016 oligos that have assigned barcodes.